Map Kinase and PI3 Kinase Pathways Regulate BDNF-Medicated Muller Cell Survival

Abstract

Abstract: : Purpose: Previous studies in our laboratory showed glutamate treatment stimulated the expression and receptor-binding of brain-derived neurotrophic factor (BDNF) in cultured Müller cells. We have hypothesized that the BDNF-receptor binding may activate signaling pathways that protect Müller cells from glutamate toxicity. In the following study, we investigated the mechanisms involved in BDNF-mediated protection of Müller cells. Methods: Cultured Müller cells were treated with L-glutamate (0.1-1.0 mM) or BDNF (50-100 ng/ml) for 48 hrs and assayed for expression of anti-apoptotic protein Bcl2 and phosphorylation of MAP kinase using immunoblots. Müller cells were also treated with BDNF or glutamate in the presence or absence of LY294002 (10-50 nM), a PI3 kinase/Akt inhibitor, or PD98059 (10-50 nM), a MEK inhibitor, and assayed for cell survival using MTS assay or fluorescent dyes calcein-AM or ethidium homodimer. Results: Glutamate or BDNF treatment of cultured rat Müller cells promoted increased phospho-MAPK and Bcl2 levels in immunoblots. Surprisingly, incubation of Müller cells with LY294002 (10-50 nM) or PD98059 (10-50 nM) resulted in ∼30-60% decrease in the number of Müller cells. In the presence of the kinase inhibitors LY294002 or PD98059, treatment of Müller cells with BDNF (50-100 ng/ml) or glutamate (1 mM) resulted in ∼40% increase in Müller cell survival as compared with cells treated with either inhibitor alone. Conclusion: Our study shows that constitutive expression and activity of the MAP kinase and PI3 kinase pathways may be necessary for Müller cell survival. Moreover, the protective effects of BDNF on Müller cells may be mediated by the MAP kinase and PI3 kinase pathways.

Keywords: 580 signal transduction • 479 Muller cells • 423 growth factors/growth factor receptors